Salsolinol (6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoneline;SAL) is a natural derivative of dopamine. It has been reported that alcohol consumption increases the level of SAL. While R-SAL is formed from enzymatic condensation of dopamine with pyruvic acid, non-enzymatic condensation of dopamine with acetaldehyde, the oxidation product of ethanol, generates the racemic mixture of (R/S)-SAL in vivo. To test a possibility of SAL representing a marker for alcohol addiction, a reliable method for quantification of the (S)-SAL stereo isomer from biological matrices is required. To improve the sensitivity and stability of the SAL stereoisomer analysis, a new approach using pentafluorobenzyl (PFB) derivatization has been explored. By optimizing derivatization procedures, SAL was derivatized fully at hydroxyl and the secondary amine groups and analyzed by electrospray ionization mass spectrometry in conjunction with chiral HPLC. [unreadable] [unreadable] During this report period, we completed development of a new quantitative method to determine enantiomeric (R/S)-salsolinol in human plasma using electrospray ionization-tandem mass spectrometry coupled to chemical derivatization and chiral separation. Salsolinol fractionated from plasma using a phenylboronic acid solid phase cartridge was derivatized with pentafluorobenzyl bromide. Resulting derivatives were base-line separated on a Chiralpak AD-H column and determined by selected reaction monitoring of the ring cleavage (mass transition from m/z 720 to 210) in the presence of deuterium labeled internal standards (mass transition from m/z 724 to 210 for d4-salsolinol). A linear response was observed for each enantiomer over the 0.5-500pg range, with correlation coefficients >0.999. The recovery of spiked salsolinol (2ng/mL) in human plasma was 50-60%. The mass spectrometric detection limit after chromatographic separation was 8fg for each isomer while the limit of detection assessed for the entire process was 5pg. The concentrations in healthy, human plasma (n=10) were in the range from 46 to 1007pg/mL for (S)-salsolinol and from 63 to 1348pg/mL for (R)-salsolinol. At these physiologically relevant concentrations, both coefficient of variance and error were less than 8% for both inter- and intra-day measurements (n=6) indicating that precision and accuracy were acceptable for reliable determination of human plasma samples. This assay method is currently applied to investigate the effect of alcohol on SAL levels and their enantiomeric distribution in human plasma.